CN115804791A - Methods and pharmaceutical compositions for treating ILK signaling pathway related diseases with mesenchymal stem cell-derived exosomes - Google Patents
Methods and pharmaceutical compositions for treating ILK signaling pathway related diseases with mesenchymal stem cell-derived exosomes Download PDFInfo
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Abstract
The invention provides a pharmaceutical composition for treating ILK signaling pathway related diseases, which contains exosomes derived from mesenchymal stem cells, particularly mesenchymal stem cells derived from pluripotent stem cells. The invention also provides methods of treating ILK signaling pathway related diseases using the exosomes.
Description
Priority of the chinese patent application entitled "method and pharmaceutical composition for treating ILK signaling pathway-related diseases by mesenchymal stem cell-derived exosomes" filed on 9/15/2021, application No. 202111078183.5, the entire contents of which are incorporated herein by reference.
Technical Field
The present invention relates to the fields of cytology and pharmacology. Specifically, the invention provides a method for treating ILK signaling pathway related diseases by using mesenchymal stem cell-derived exosomes and a pharmaceutical composition for treating ILK signaling pathway related diseases.
Background
Integrin linked kinase (ILK, otherwise known as p59 ILK) was originally identified from a two-hybrid screen of a human placental cDNA library by its ability to bind and phosphorylate the B1-Integrin cytoplasmic domain (Hannigan et al, nature, 1996). In the study of ILK, it was found that overexpression of ILK results in disruption of the epithelial morphology of IEC18 cells and reduced adhesion of cells to the extracellular matrix. Studies have also found a role for ILK in activating transcription in the Wnt signaling cascade (Novak et al, proc.natl.acad.sci.usa, 1998). The activity of ILK has been shown to be regulated in other signaling pathways, including those involving G-protein (Tu et al, mol. Cell. Biol., 1999), phosphatidylinositol 3-kinase, protein kinase B, and glycogen synthase kinase 3 (Delcommenne et al, proc. Natl. Acad. Sci. U.s.a., 1998). ILK signaling pathways are also known in the art to be associated with angiogenesis, which is important for physiological and pathological angiogenesis. Endothelial ILK plays a key role in vascular development and Endothelial Cell (EC) survival through integrin-matrix interactions.
Mesenchymal stem cells are important members of the stem cell family. Natural mesenchymal stem cells are derived from the early developmental mesoderm. Mesenchymal stem cells can be isolated from various tissues and cultured. Mesenchymal stem cells can also be differentiated from universal stem cells. Among them, induced pluripotent stem cells (ipscs), also called induced pluripotent stem cells or artificial pluripotent stem cells, are dry cells having embryonic stem cells that are artificially prepared. The induced pluripotent stem cells were first produced by Shinya Yamanaka, a scientist of Japan, in 2006, and were one cell type similar to embryonic stem cells and embryonic APSC pluripotent cells obtained by transferring a combination of four transcription factors (Oct 4, sox2, klf4, and c-Myc) into differentiated somatic cells using a viral vector and reprogramming the cells. The induction of universal stem cells has been widely used in the fields of biotechnology and medical research.
Recent findings around the function of extracellular vesicles (EVs, small particles of 40-100 nm) secreted by stem cells have led to significant advances in regenerative medicine. The regenerative effects of stem cell transplantation therapy are somewhat benefited by the paracrine effects of EV release. EVs do not contain MHC I or MHC II proteins, do not increase the risk of immunogenicity, and are not tumorigenic, thus overcoming several disadvantages of cell transplantation therapies. It was found that exosomes from mesenchymal stem cells (MSC EVs) could help repair injured tissues (Harrell et al, 2019 tang et al, 2021. Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) have also been applied in regenerative medicine. EVs secreted by iPSC-MSCs promote wound repair (Zhang et al, 2015), angiogenesis (Hu et al, 2015), bone angiogenesis (Hu et al, 2015) and bone regeneration (Qi et al, 2016). It has been found that EV from mesenchymal stem cells can transfer functional miRNA to target cells to modulate their function, resulting in therapeutic effects in damaged tissues. For example, miR-644-5P in stem cells improves ovarian function in chemotherapy-induced ovarian injury rats by targeting P53 (Sun et al, 2019). Similarly, EV secreted by human amniotic mesenchymal stem cells releases miRNA-320a, which modulates SIRT4 to protect POI mice from ovarian oxidative stress (Ding et al, 2020 a). miRNA-17-5p derived from human umbilical cord mesenchymal stem cells improves ovarian function after chemotherapy by modulating SIRT7 (Ding et al, 2020 b). Exosomes produced by bone marrow mesenchymal stem cells can target PTEN by delivering mir-144-5p, thereby improving ovarian function in chemotherapy-induced ovarian dysfunction rats (poplar et al, 2020). However, these studies also found that there were significant differences in the roles, mechanisms of action, and involved pathway members (proteins, genes or their regulators, etc.) of stem cells of different origins and EVs secreted therefrom in target cells.
There is also a need in the art to investigate the effects of stem cells or other cell exosomes of various origins on cells, including the effects on integrin linked kinase, the ILK pathway, and their effects in the treatment of diseases associated with the ILK signaling pathway, thereby providing novel therapies and drugs that are effective and safe for the treatment of diseases associated with the ILK signaling pathway.
Disclosure of Invention
The present application provides novel therapies and pharmaceutical compositions for treating ILK-related diseases using mesenchymal stem cell-derived exosomes. The inventor of the applicant firstly discovers that the exosome derived from the mesenchymal stem cell has the function of regulating the expression of the protein related to the ILK signal pathway of the cell, including ILK and the like, and can be used for treating the disease related to the ILK.
In one aspect of the invention, there is provided a method of modulating the ILK signaling pathway in a cell comprising applying mesenchymal stem cell-derived exosomes to cells in tissue or culture medium.
In the present invention, the term mesenchymal stem cells, also called multipotent mesenchymal cells, are mainly obtained from fat or bone marrow and are capable of differentiating into various cells of mesodermal origin, such as bone, fat, cartilage, tendon, muscle, and the like. Mesenchymal stem cells can be isolated from various tissues and cultured, but their abilities and cell surface markers differ from each other depending on their origin. Mesenchymal stem cells are generally defined by cells that can differentiate into osteocytes, chondrocytes, and myocytes, and express cell surface markers such as CD73 (+), CD105 (+), CD34 (-) and CD45 (-).
In one aspect of the present invention, the mesenchymal stem cell is a bone marrow-derived, adipose-derived, umbilical cord blood-derived, dental-derived or universal stem cell-derived mesenchymal stem cell. In yet another aspect of the present invention, the mesenchymal stem cell is a universal stem cell-derived mesenchymal stem cell.
In the present invention, the term pluripotent stem cell refers to a stem cell capable of producing all embryonic cell types. Natural pluripotent stem cells include embryonic stem cells. Induced pluripotent stem cells (ipscs), also called induced pluripotent stem cells or artificial pluripotent stem cells, are dry cells with embryonic stem cells that are artificially prepared, and can be obtained by transferring a combination of four transcription factors (Oct 4, sox2, klf4, and c-Myc) into differentiated somatic cells using a viral vector, and reprogramming the cells.
In one aspect of the invention, the mesenchymal stem cell is a human-induced universal stem cell-derived mesenchymal stem cell.
Extracellular vesicles are membrane vesicles secreted by cells. The extracellular vesicles may have a diameter (referring to their largest dimension in the case of particles that are not spheres) of between about 10nm to about 5000 nm. In the present invention, exosomes refer to small secretory vesicles, typically having a diameter (in the case of particles that are not spheres, their largest dimension) between about 30nm to about 250nm, for example having a diameter between about 30nm to about 200 nm. Exosomes comprise, or have in their membranes, nucleic acids, proteins, or other biomolecules and may serve as carriers between different locations in the body or biological system.
Exosomes may be isolated from a variety of biological sources including mammals such as mice, rats, guinea pigs, rabbits, dogs, cats, cows, horses, goats, sheep, primates or humans. Exosomes may be isolated from biological fluids such as serum, plasma, whole blood, urine, saliva, breast milk, tears, sweat, synovial fluid, cerebrospinal fluid, semen, vaginal fluid, ascites fluid, and amniotic fluid. Exosomes may also be isolated from experimental samples such as culture media taken from cultured cells.
In one aspect of the present invention, there is provided a method for modulating the ILK signaling pathway in cultured cells, comprising adding to a cell culture broth an exosome inducing pluripotent stem cell-derived mesenchymal stem cells. In one aspect of the present invention, the mesenchymal stem cells are cells passaged 1 to 15 times, preferably 1 to 10 times, and most preferably 3 to 7 times.
In one aspect of the invention, the exosomes are added to the culture broth in an amount of about 1-500ug/ml, preferably about 5-250ug/ml, more preferably about 10-200ug/ml.
In one aspect of the invention, the cell is one in which the ILK pathway (e.g., the PTEN/ILK/AKT pathway) is aberrant (down-regulated). In yet another aspect of the invention, the method restores ILK pathway activity in the cell, e.g., by upregulating ILK activity in the cell.
In one aspect of the invention, the method includes the step of detecting ILK pathway-related genes (e.g., PTEN/ILK/AKT pathway) such as Ilk, pten, krt18, ccnd1, cdkn2a, vegfa, ptgs 2.
In one aspect of the present invention, there is provided a method for treating an ILK signaling pathway-related disease, comprising administering to a patient a universal stem cell-derived mesenchymal stem cell-derived exosome. In one aspect of the invention, the application of the mesenchymal stem cell-derived exosome in preparing a medicament for treating ILK-related diseases is also provided.
In one aspect of the present invention, there is provided a pharmaceutical composition for treating ILK-related diseases, which contains mesenchymal stem cell-derived exosomes.
In one aspect of the present invention, there is provided a use of a mesenchymal stem cell-derived exosome in a pharmaceutical composition for treating an ILK-related disease.
ILK-related diseases, also referred to herein as ILK signaling pathway-related diseases, include diseases or disorders associated with altered ILK expression and/or activity, including diseases or disorders responsive to modulation of ILK expression. The role of ILK in activating transcription in the Wnt signaling cascade is known in the art. ILK is also known to play a role in other signaling pathways, including those involving G-protein, phosphatidylinositol 3-kinase, protein kinase B, and glycogen synthase kinase 3. Upstream regulatory signals of ILK include, for example, PTEN (a lipid phosphatase that negatively regulates ILK activation), and the like. Known ILK signal paths also include downstream AKT paths, and the like. In one aspect of the invention, the ILK signal pathway comprises the PTEN/ILK/AKT pathway.
ILK signaling pathways are known in the art to be associated with angiogenesis, which is important for physiological and pathological angiogenesis. Endothelial ILK plays a key role in vascular development and Endothelial Cell (EC) survival through integrin-matrix interactions. Integrin-mediated signaling, in conjunction with Vascular Endothelial Growth Factor (VEGF) receptors, promotes morphological changes, cell proliferation and motility in endothelial cells. Thus, ILK signaling pathway-related diseases include diseases associated with aberrant or pathological angiogenesis, such as, but not limited to, various cancers, psoriasis, and age-related macular degeneration.
Diseases associated with abnormal or pathological angiogenesis include cancers such as brain cancer, esophageal cancer, bladder cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, pancreatic cancer, head and neck cancer, prostate cancer, thyroid cancer, renal cancer, and ovarian cancer, melanoma, lymphoma, glioma, glioblastoma, and any other cancer disease, among others. In this regard, ILK signaling pathway related diseases also include heart diseases (e.g., cardiomyopathy, cardiovascular disease, congenital heart disease, coronary heart disease, heart failure, hypertensive heart disease, inflammatory heart disease, valvular heart disease).
ILK signaling pathway-related diseases also include a variety of known metabolic disorders including inherited metabolic disorders. Such metabolic disorders include, but are not limited to, diabetes, hyperlipidemia, lactic acidosis, phenylketonuria, tyrosinemia, urea cycle disorders, and the like.
In addition, it is known in the art that the ILK signaling pathway is closely related to inflammation: leukocyte extravasation is an important step in inflammation, where integrins have been shown to play an important role by mediating leukocyte interactions with vascular endothelium and the underlying extracellular matrix. As a link between integrins and cytoskeletal systems, ILK is a key molecule involved in cell-cell and cell-matrix interactions.
In this regard, ILK signaling pathway related diseases include various inflammatory diseases such as, but not limited to, asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, ankylosing spondylitis, reiter's syndrome, crohn's disease, ulcerative colitis, systemic lupus erythematosus, psoriasis, atherosclerosis, rheumatoid arthritis, osteoarthritis, or multiple sclerosis.
It has been found that the AKT pathway downstream of ILK is critical for folliculogenesis, and disruption of the AKT pathway impairs primordial follicle survival, leading to the development of POI (Kalich-Philoph et al, 2013 Wang et al, 2019). In this respect, diseases related to the ILK signaling pathway also include ovarian-related reproductive disorders, such as reduced ovarian function caused by chemotherapy, diminished Ovarian Reserve (DOR), premature Ovarian Insufficiency (POI), premature Ovarian Failure (POF), and the like, particularly Premature Ovarian Failure (POF).
In one aspect of the present invention, the mesenchymal stem cell used for exosome extraction in the aforementioned method and pharmaceutical composition is a mesenchymal stem cell obtained after subculturing a mesenchymal stem cell differentiated and formed from induced pluripotent stem cells and the like. For example, the mesenchymal stem cell is a cell passaged 1 to 15 times, preferably a cell passaged 1 to 10 times, and most preferably a cell passaged 3 to 7 times. In the present invention, the primary generation cells for subculture, i.e., P0 cells, generally refer to mesenchymal stem cells in which the mesenchymal stem cell-derived cells appear earliest after being induced and differentiated, i.e., cells having mesenchymal stem cell characteristics (e.g., having mesenchymal stem cell-specific surface markers, etc.) appear in a cell population that account for 50% or more, or preferably 75% or more, or more preferably more than 90% of the total number of cells. The primary generation cells can be directly used for subculture, and can also be used for subculture after being recovered after being frozen and preserved. The inventor of the present application unexpectedly found that the exosome extracted from the mesenchymal stem cell induced by the differentiation of the pluripotent stem cell and the like has repairing activity on the cell of the damaged tissue (such as the egg cell and the granulosa cell in each stage of the ovary). The inventors of the present application found that, among the mesenchymal stem cells subjected to subculture, the repair activity of exosomes produced by mesenchymal stem cells of earlier generations (such as cells subjected to 15 or less passages, particularly cells subjected to 7 or less passages) on cells of damaged tissues (such as egg cells and granulosa cells at various stages in the ovary) is significantly superior to the activity of exosomes produced by mesenchymal stem cells of later generations (such as cells subjected to 10-15 or more passages). Meanwhile, the inventors of the present application have unexpectedly found that the chance of successfully obtaining exosomes having the activity of repairing damaged tissues from mesenchymal stem cells formed by inducing differentiation of pluripotent stem cells or the like is greatly increased relative to mesenchymal stem cells isolated from tissues (e.g., bone, fat, cartilage, umbilical cord), one of which is represented in that the difference between the number and quality of exosomes collected from mesenchymal stem cells cultured by 15 or less passages and the level of repair activity of cells of damaged tissues (e.g., egg cells and granular cells at each stage in ovary, etc.) detected in different production batches can be maintained substantially within 20%. And the difference in the number and repair activity of about 10 subcultures or the like using exosomes collected from mesenchymal stem cells isolated from fat or umbilical cord is about 50% or more.
Exosomes for use in the present invention may be performed by various methods known in the art. Methods for isolating exosomes include ultrafiltration, polymer precipitation, size chromatography, or ultracentrifugation, among others. Preferably, the exosome used in the present invention may be prepared by ultrafiltration.
In one aspect of the invention, the exosomes for use in the invention are prepared from cell culture fluid by ultrafiltration. In the ultrafiltration method used in the present invention, exosomes are screened using an ultrafiltration membrane having a molecular weight cut-off of about 100 kDa. Exosomes are present in components that are unable to pass an ultrafiltration membrane with a molecular weight cutoff of about 100 kDa.
In the ultrafiltration process used in the present invention, it is also possible to use a multiple filtration system and process, i.e. filtration step by step through filters of different pore sizes. In yet another aspect of the invention, the ultrafiltration method further comprises the step of filtering with a filter having a pore size of 4 μm and/or a filter having a pore size of 0.22 μm prior to the ultrafiltration membrane having a molecular weight cut-off of about 100 kDa. For example, filtration can be carried out stepwise through a cell filter of 4 μm pore size, a filter of 0.22 μm pore size and a filter having an MWCO (molecular weight cut-off) of 100 kD.
The pharmaceutical composition for preventing or treating diseases related to ILK signaling pathway provided by the invention contains the above exosome in pharmaceutically effective amount. The exosomes may be comprised in the pharmaceutical composition alone or together with one or more pharmaceutically acceptable carriers, excipients or diluents. By pharmaceutically effective amount is meant an amount sufficient to prevent, ameliorate or treat the symptoms of a disease associated with the ILK signaling pathway.
In one aspect of the invention, the dosage of the exosome in the pharmaceutical composition provided by the invention is about 1-500ug, preferably about 5-250ug, more preferably about 10-200ug. In yet another aspect of the invention, the exosomes are dosed at about 40-5000ug/kg body weight, preferably about 400-4000ug/kg body weight.
In one aspect of the invention, the pharmaceutical composition provided herein comprises a dosage of said exosomes of about 1 x 10 9 To 1X 10 12 Preferably about 1X 10 10 To 1X 10 11 And (4) respectively. In yet another aspect of the invention, the exosome is dosed at about 1 x 10 10 To 4X 10 13 Preferably about one/kg body weight1×10 11 To 4X 10 12 One/kg body weight.
In addition, the pharmaceutical compositions provided herein can be prepared as unit dosage formulations suitable for administration to a patient according to methods conventional in the pharmaceutical arts, and such formulations contain an amount effective for administration by one or several administrations. The pharmaceutical compositions provided herein may be in a single or multiple administration dosage form. The pharmaceutically effective amount may be suitably changed depending on the severity of the disorder, age, body weight, health condition and sex of the patient, administration route, treatment period and the like.
The exosomes employed in the pharmaceutical compositions provided by the present invention are particularly suitable for use in therapy by multiple administrations. In one aspect, the pharmaceutical compositions provided herein are in the form of a multi-dose dosage form. In yet another aspect of the present invention, the pharmaceutical compositions provided herein are in dosage forms that are administered at intervals of about 1 day to about 7 days. Preferably, the pharmaceutical compositions are in dosage forms that are administered about 2-7 days apart.
In one aspect, the present invention provides a pharmaceutical composition in a dosage form, in particular in said multiple administration dosage form, wherein each administration dose of said exosome is about 1-500ug, preferably about 5-250ug, more preferably about 10-200ug. In another aspect of the invention, the pharmaceutical composition provided by the invention is in a dosage form, in particular in said multi-administration dosage form, wherein each administration dose of said exosomes is about 40-5000ug/kg body weight, preferably about 400-4000ug/kg body weight.
In one aspect, the present invention provides a pharmaceutical composition in a dosage form, in particular in said multi-dose dosage form, wherein each dose of said exosome is about 1 x 10 9 To 1X 10 12 Preferably about 1X 10 10 To 1X 10 1 1 piece. In another aspect of the present invention, the pharmaceutical composition provided by the present invention, in particular in said multi-administration dosage form, each administration dose of said exosome is about 1 × 10 10 To 4X 10 13 Preferably about 1X 10, per kg body weight 11 To 4X 10 12 One/kg body weight.
The pharmaceutical compositions provided by the present invention are physiologically acceptable and do not generally cause allergic reactions, such as gastrointestinal disorders or dizziness or the like, when administered to a human. Examples of the carrier, excipient and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, bulking agent, deflocculant, lubricant, humectant, edible essence, emulsifier, antiseptic, etc. can be added.
In addition to the active ingredient, the pharmaceutical preparations may contain one or more pharmaceutically acceptable usual inert carriers such as preservatives, analgesics, solubilizers, stabilizers and the like for injection or bases, excipients, lubricants or preservatives and the like for topical preparations.
The composition or pharmaceutical preparation of the present disclosure prepared as described above can be administered to mammals, such as rats, mice, livestock, humans, etc., through various routes including parenteral and oral routes. Any mode of administration commonly used in the art may be used.
The present invention also provides a method of treating an ILK-related disease comprising administering mesenchymal stem cell-derived exosomes to a patient. The patient may be a mammal, e.g., rat, mouse, livestock, human, etc. Any mode of administration commonly used in the art may be used.
In one aspect of the present invention, the mesenchymal stem cell in the method for treating an ILK-related disease is a bone marrow-derived, adipose-derived, umbilical cord blood-derived, dental-derived or universal stem cell-derived mesenchymal stem cell. In one aspect of the present invention, the mesenchymal stem cell in the method for treating an ILK signaling pathway-related disease is an induced pluripotent stem cell-derived mesenchymal stem cell.
In one aspect of the present invention, the mesenchymal stem cell in the method for treating ILK-related diseases is a cell passaged 1 to 10 times, preferably a cell passaged 3 to 7 times.
In one aspect of the invention, in the method for treating ILK-related diseases, the exosomes are prepared by an ultrafiltration method, and the exosomes are screened by an ultrafiltration membrane with the molecular weight cutoff of about 100 kDa. Preferably, the ultrafiltration method further comprises the step of filtering with a filter with a pore size of 4 μm and/or a filter with a pore size of 0.22 μm before the ultrafiltration membrane with a molecular weight cut-off of about 100 kDa.
In one aspect of the present invention, the method for treating an ILK-related disease comprises administering about 1-500ug, preferably about 5-250ug, and more preferably about 10-200ug of exosomes to a patient. In yet another aspect of the present invention, the method for treating an ILK signaling pathway-related disease comprises administering to a patient exosomes at 40-5000ug/kg body weight, preferably at about 400-4000ug/kg body weight.
In one aspect of the invention, the method of treating an ILK-related disorder comprises administering to the subject about 1X 10 9 To 1 × 10 12 Preferably about 1X 10 10 To 1X 10 11 And (4) one exosome. In still another aspect of the present invention, the method for treating an ILK signaling pathway related disease comprises administering to a patient a 1X 10 10 To 4X 10 13 Preferably about 1X 10/kg body weight 11 To 4X 10 12 Exosomes per kg body weight.
In one aspect of the present invention, the exosome is administered to the patient one or more times in the method for treating an ILK-associated disease.
In one aspect of the present invention, the exosome is administered to the patient at intervals of about 1 day to 7 days, preferably at intervals of about 2 days to 7 days, in the method for treating an ILK-associated disease.
In one aspect of the present invention, in the method for treating ILK-related diseases, particularly in the multi-dose treatment method, the dosage of each administration of the exosome is about 1-500ug, preferably about 5-250ug, more preferably about 10-200ug. In another aspect of the present invention, the present invention provides said method for treating an ILK signaling pathway-related disease, particularly said method for multiple administration, wherein each administration dose of said exosome is about 40-5000ug/kg body weight, preferably about 400-4000ug/kg body weight.
In one aspect of the present invention, in the method for treating an ILK-related disease, the exosome is administered at a dose of about 1 × 10 per administration 9 To 1 × 10 12 Preferably about 1X 10 10 To 1 × 10 11 And (4) respectively. In another aspect of the present invention, the present invention provides said method for treating a disease associated with the ILK signaling pathway, particularly said method for multiple administration, wherein each administration dose is about 1 × 10 10 To 4X 10 13 Preferably about 1X 10/kg body weight 11 To 4X 10 12 One per kg body weight.
In one aspect of the invention, the method of treating an ILK-related disease is a method of treating a disease associated with abnormal or pathological angiogenesis, such as cancer, heart disease, metabolic disorder, inflammatory disease, or ovarian-related reproductive disorder.
Drawings
FIG. 1 shows the results of identifying exosomes (iPSC-MSCs-EVs) obtained from human induced pluripotent stem cells (iPSC-MSCs). The iPSC-MSCs-EVs were characterized by immunoblotting (fig. 1A), transmission electron microscopy (fig. 1B), and RNA distribution analysis (fig. 1C), respectively.
FIG. 2 shows that iPSC-MSCs-Evs modulate ILK signaling pathway in chemotherapy injury model granulosa cells. Figure 2A shows a RNA sequencing differential gene volcano plot. Figure 2B shows a graph showing the results of IPA software analysis. FIG. 2C shows the difference in gene change analyzed by the Qlucore software. FIG. 2D is a heat map analysis of ILK signaling pathway-related differential genes. FIG. 2E is a qPCR result analysis of ILK signal pathway-related expression difference genes.
FIG. 3 shows that iPSC-MSCs-EVs can reverse PTEN/ILK/AKT pathway downregulation by CTX in vivo or in vitro experiments. FIG. 3A is a graph showing the results of immunoblotting for detecting the expression of ILK signaling pathway-related proteins in granulosa cells. FIG. 3B is a graph showing the results of immunoblotting to detect the expression of ILK signal pathway-related proteins in vitro cultured ovaries. FIG. 3C is a graph showing the results of immunohistochemical detection of ILK protein expression in the ovaries of in vitro culture and in the ovaries of adult mice.
FIG. 4 is a graph showing the results of iPSC-MSCs-EVs treatment of CTX-induced apoptotic granulosa cells. FIG. 4A is a graph showing the results of MTS assay. FIG. 4B is a graph showing the results of immunoblotting to detect the expression of apoptosis markers in granulosa cells.
FIG. 5 is a graph showing the results of the effect of co-culture of iPSC-MSCs-EVs with 4 HC-CTX-treated ovaries. Fig. 5A is a graph showing the results of follicle counting. FIG. 5B is a graph showing the results of immunohistochemistry experiments.
FIG. 6 is a graph showing the results of the effect on ovaries of mice treated with CTX and co-cultured with iPSC-MSCs-EVs. FIG. 6A is a graph showing the results of HE staining experiments. Fig. 6B is a graph showing the result of follicle counting. FIG. 6C is a graph showing the results of immunohistochemical detection of apoptotic-grade proliferation-associated proteins.
Detailed Description
The spirit and advantages of the present invention will be further illustrated by the following examples, which are provided by way of illustration and are not intended to be limiting.
Example 1 isolation and characterization of exosomes of mesenchymal stem cells of human induced pluripotent stem cell differentiation
Induction of Induced Pluripotent Stem Cells (iPSCs)
Using NEPA21 electrotransfer instrument to convert 10 into 6 A mixture of Human Dermal fibroblasts (ATCC # PCS-201-010) and 3. Mu.g of plasmids (UL (addendum # 27080), OP (addendum # 27077) and SK (addendum # 27078)) was electroporated. The cells thus transfected were seeded on a plate coated with matrigel, and after 24 to 29 days of incubation with mTeSR (STEMCELL # 85850), dryness of the cells was evaluated against Alkaline Phosphatase (AP) staining to determine whether or not introduction of a specific transcription factor reprograms terminally differentiated somatic cells into pluripotent stem cells.
Induction of iPSC-MSCs (induced multiplexed stem-derived sensed stem cells)
Inducing the human iPSCs obtained in the previous step into Mesenchymal stem cells (iPSC-MSCs) for inducing differentiation of the human induced pluripotent stem cells by adopting an interstitial Progenitor Kit (Mesenchymal Progenitor Kit) according to the instruction method of the instruction book of the Kit
Separation of iPSC-MSCs exosomes
Cell supernatants from 3 rd to 7 th generation cultured iPSC-MSCs were collected and Exosomes (EVs) were isolated therefrom using ultrafiltration (Milipore, 100 kDa).
Experimental materials: refrigerated centrifuge (Eppendorf 5804R),
Ultra-15Centrifugal Filter Unit(Millipore，100KD#UFC910096)。
the main experimental steps of the ultrafiltration method are as follows:
1) The collected cell supernatant was centrifuged at 300g at 4 ℃ for 1Omin to remove residual cells;
2) Centrifuging at 2000g for 20min to remove cell debris;
3) The supernatant was carefully collected and filtered through a 0.22 μm pore size filter;
4) Adding the collected supernatant to
In Ultra-15, 3000g is centrifuged;
5) After the supernatant is ultrafiltered, adding PBS and ultrafiltering again, repeating the washing for 2 times, and finally dissolving the exosome remained on the filter membrane by using 200 mu l of PBS to obtain an exosome solution.
The exosomes produced by the MSC are identified and observed, including the observation of EV morphology and size by using an electron microscope, and the identification of the expression of EV surface markers CD9, CD63 and CD81 by using a western blotting technology.
FIG. 1 shows the results of identifying exosomes (iPSC-MSCs-EVs) obtained from human induced pluripotent stem cells (iPSC-MSCs).
The iPSC-MSCs-EVs were characterized by immunoblotting, transmission Electron Microscopy (TEM), and RNA profiling analysis, respectively, as shown in figure 1. Immunoblotting (fig. 1A) showed that the isolated exosomes were positive for the exosome surface markers CD63, CD9, CD81 and Hsp70, but negative for the exosome negative marker Calnexin. By TEM (FIG. 1B), iPSC-MSCs-EVs showed a typical round cup-shaped exosome structure with a diameter of about 40-150 nm. In addition, the distribution of RNA extracted from iPSC-MSCs-EVs was examined by Agilent 2100Bioanalyzer (FIG. 1C), and it was shown that the peak of RNA was concentrated between 20-200nt, which is significantly different from the size distribution of RNA in cells.
The identification characteristics show that exosome is extracted from the supernatant of iPSC-MSCs cells.
Example 2 in vitro experiments to verify that iPSC-MSCs-EV treatment can modulate ILK signaling pathway in granulosa cells
Changes in the ILK signaling pathway were observed by preparing granulosa cells that mimic chemotherapy injury and subjecting the injured granulosa cells to treatment with exosomes in coculture.
Granulosa cells from mice born for 20 to 23 days were isolated and divided into a control group, a Cyclophosphamide (CTX) apoptosis-inducing group, and a cyclophosphamide and exosome (CTX-EVs) co-treatment group. Wherein CTX is added into the cell culture solution according to the concentration of 2mg/ml, and exosome (iPSC-MSCs-EVs) is added into the cell culture solution according to the concentration of 20 mu g/ml. Exosome concentration using protein total amount measurement kit Pierce TM The BCA Protein Assay Kit was used for the Assay. After 24 hours of incubation, the next assay was performed.
After the in vitro experiment treatment is carried out on the experimental group granular cells, RNA is extracted by TRIzol, and a BGISEQ-500 system (Huada gene, shenzhen, china) is adopted for RNA sequencing of transcriptome. As a result of sequencing, 1451 Differential Expression Genes (DEG) were found in the CTX-treated group compared with the CTX-EVs group (FIG. 2A). KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis shows that the expression difference Genes are mainly concentrated on a cell signal transfer and cell metabolism change pathway; IPA software analysis showed that the ILK signal path was suppressed in the CTX processing group and reactivated in the CTX-EV group (fig. 2B).
Further gene screening was performed using Qlucore software, selecting 1000 genes with the greatest differences among genes whose expression fold difference was greater than 1.5 and p value was less than 1.5e (fig. 2C), and then analyzing these genes using IPA software, showed that the ILK signaling pathway was one of the most significant pathways. Thermographic analysis of ILK signaling pathway-associated differential genes further confirmed that these differential genes were down-regulated in the CTX-treated group and up-regulated in the CTX-EV group compared to the control group (fig. 2D).
Further, ILK-related genes (including Ilk, pten, krt18, ccnd1, cdkn2a, vegfa, ptgs 2) were analyzed by QPCR and confirmed to have the above-mentioned changes.
QPCR was performed using QuantStaudio 7Flex Real-time PCR System (Applied Biosystems) and SYBR Green Master Premix Ex Taq (Takara). Wherein the primers used include: .
ILK: forward, 5 'GAACGACCTCAATCAGGGGGG-3'; reverse, 5 'CATTAATCCGTGCTCCACGC-3';
bcl2: forward, 5 'GAACTGGGGGAGGATGTGTGG-3'; reverse, 5 'GCATGCTGGGGCCATATAGT-3';
bax: forward, 5 'TGAAGACAGGGGCCTTTTTTTG-3'; reverse, 5 'AATTCGCCGGAGACACTCG-containing 3';
krt18: forward, 5 'ACCACCAAGTCTGCCGAAAT-3'; reverse, 5 'CCGAGGCTGTTTCTCCAAGTT-3';
ccnd1: forward, 5 'CAACTTCCTCTCCTGCTTACCG-3'; reverse, 5 'GATGGAGGGGTCCTTGTTTTAG-3';
vegfa: forward, 5 'GCACATAGAGAGAGAATGAGCTTCT-3'; reverse, 5 'CTCCGCTCTGAACAGGCT-3';
ptgs2: forward, 5 'CATCCCTTGCGAAGTT-3'; reverse, 5 'CATGGGAGTTGGGCAGTCAT-3';
cdkn2a: forward, 5 'CGCTTCTCACCTCGCTTGT-3'; reverse, 5' AGTGACCAAGAACCTGCGAC-;
pten: forward, 5' TGGATTCGACTTAGACTTGACCT-; reverse, 5 'GCGGTGTCATAATGTCTCTCAG-3';
gapdh-: forward, 5 'GAGAGTGTTCCTCGTCCCG-3'; reverse, 5 'ACTGTGCCGTTGAATTTGCC-3'.
Normalization was performed with rat-Gapdh as the internal control. Relative mRNA expression levels were determined using a 2-. DELTA.Ct method.
The results are shown in fig. 2E, where Ptgs2 (promoting cumulus granulosa cell survival during expansion) was down-regulated in the CTX-treated group, but significantly up-regulated in the CTX plus EV-treated group (p < 0.05), compared to the control group. The expression of the apoptotic gene Bax in the CTX group was significantly increased (p < 0.05), but significantly decreased in the CTX plus EV group (p < 0.05). The anti-apoptotic gene Bcl2 did not differ significantly between the groups. High throughput miRNA sequencing showed that 9 of the first 50 micrornas (mirnas) expressed in iPSC-MSC-EV target PTEN, a lipid phosphatase that negatively regulates ILK activation. These results show that iPSC-MSC EVs protect granulosa cells from apoptosis and maintain normal function by modulating the ILK signaling pathway, for example by transferring functional miRNAs to reverse CTX-induced down-regulation of ILK pathway in granulosa cells.
Example 3iPSC-MSCs-EVs reverse PTEN/ILK/AKT pathway Down-Regulation in vivo or in vitro experiments
Further analyzing the specific expression condition of the ILK signal channel related protein, and carrying out immunoblotting detection on the protein of the particle cells of the experimental group. As the results (fig. 3A) show, the expression of ILK in the CTX group was lower than that in the control group, and the expression of ILK in the CTX-EV group was significantly increased. Meanwhile, expression of PTEN is obviously up-regulated after CTX treatment, but is inhibited after the addition of iPSC-MSCs-EVs. The expression of ILK was significantly reduced after CTX treatment, but was significantly increased after the addition of iPSC-MSCs-EVs. The p-AKT/AKT ratio decreased significantly after CTX treatment, but this decrease was significantly suppressed after the addition of iPSC-MSC-EVs. This means that CTX treatment suppressed the ILK/AKT pathway by up-regulating PTEN expression, but iPSC-MSCs-EVs treatment could reverse this effect.
As the ILK pathway is also involved in regulating the G1/S/G2 phase of the cell cycle, the cell cycle related proteins Ccnb1, ccnd1 and P27 are simultaneously detected in the experiment. The results show that the expression of P27 is significantly increased after the addition of chemotherapeutic drugs, but the expression of P27 is decreased after the addition of iPSC-MSCs-EVs. The expression of Ccnd1 is significantly reduced after the addition of chemotherapeutic drugs, but is significantly increased by the addition of iPSC-MSCs-EVs. Ccnb1 did not change significantly (FIG. 3B). Western Blot is used for detecting the change of ilk channel expression in ovaries cultured in vitro, and the result shows that the expression of PTEN can be obviously inhibited after iPSC-MSCs-EVs are added. The expression of IIK is reduced obviously after the addition of chemotherapeutic drugs, but the expression is increased obviously after the addition of iPSC-MSCs-EVs. The p-AKT/AKT ratio decreased significantly after addition of the chemotherapeutic drugs, but this decrease was significantly inhibited by the addition of iPSC-MSCs-EVs (FIG. 3C).
Expression of ILK in the ovaries was detected by IHC staining of adult mouse ovaries and ovaries cultured in vitro. The results showed that the expression of ILK was decreased after chemotherapy treatment, but increased after iPSC-MSCs-EVs transplantation in adult mice. The results of the in vitro ovarian culture were consistent with those of adult mice (fig. 3D). These results show that iPSC-MSCs-EVs reversed PTEN/ILK/AKT pathway down-regulation by CTX in vitro and in vivo experiments.
Example 4 in vitro experiments prove that iPSC-MSCs-EVs regulate ILK signal pathway to inhibit apoptosis of granulosa cells
Granulosa Cells (GCs) from 20 to 23 day old mice were isolated and divided into a control group, a Cyclophosphamide (CTX) induced apoptosis group, and a CTX and iPSC-MSCs-EVs co-treated group.
CTX was added to the cell culture broth at a concentration of 4 mg/ml. The iPSC-MSCs-EVs were added to the cell culture solution at a concentration of 2. Mu.g/ml, 20. Mu.g/ml or 100. Mu.g/ml. After 24 hours of incubation, the next assay was performed.
FIG. 4 is a graph showing the results of iPSC-MSCs-EVs treatment of CTX-induced apoptotic granulosa cells. The results showed that iPSC-MSCs-EVs could greatly improve cell survival in a dose-dependent manner compared to CTX-treated group by MTS assay (fig. 4A). Immunoblot results (fig. 4B) showed that CTX significantly increased the expression of the apoptosis marker clear Caspase 3 in granulosa cells. And the iPSC-MSCs-EVs treatment can obviously inhibit chemotherapy-induced granular cell apoptosis, including reducing the expression of cleared Caspase 3 and increasing the expression of cell proliferation marker PCNA in granular cells.
The results show that iPSC-MSCs-EVs treatment can inhibit apoptosis of granular cells induced by CTX and promote proliferation of granular cells by regulating ILK signal pathway.
Example 5 ovarian in vitro culture experiment proves that iPSC-MSCs-EVs regulate ILK signal channel to protect follicular development and inhibit apoptosis
Ovaries of 2.5-day-old mice were dissected and divided into control groups, mock chemotherapy groups containing 10. Mu.M 4-hydroxycyclophosphamide (4 HC-CTX), or co-treatment groups of 10. Mu.M 4HC-CTX and 100. Mu.g/ml iPSC-MSC-EVs. The culture was carried out for 72 hours.
FIG. 5 is a graph showing the results of co-culture of iPSC-MSCs-EVs and 4 HC-CTX-treated ovaries.
The results of follicle counts (FIG. 5A) show that primordial follicles from ovaries cultured with 4HC-CTX are significantly reduced compared to the control group. However, in 4HC-CTX ovaries co-treated with EVs, the number of primordial follicles was significantly increased compared to the 4HC-CTX group. The immunohistochemistry experiment result (FIG. 5B) shows that iPSC-MSC-EVs reduce the expression of cleared Caspase 3 and increase the expression of PCNA compared with the 4HC-CTX group.
The result shows that iPSC-MSC-EVs can inhibit apoptosis caused by CTX induction and promote the recovery of ovaries.
Example 6 in vivo experiments demonstrated that hiPSC-MSCs-EVs have protective effects on ovarian apoptosis in mice.
The 28-day mice were divided into treatment groups including a control group, a CTX group and a CTX-EV group. For the mice in the CTX group, CTX (120 mg/ml) was intraperitoneally injected twice a week, while 200ul of physiological saline was injected into the tail vein for six times on days 1, 3, 5, 10, 12, and 14, respectively. For the mice in the CTX-EV group, CTX (120 mg/ml) was used for intraperitoneal injection twice a week, while tail vein injection EV was performed at 200 ug/time for six times on days 1, 3, 5, 10, 12, and 14, respectively. For the mice of the control group, the same dose of physiological saline was injected.
FIG. 6 is a graph showing the results of the effect on ovaries of mice treated with CTX and co-cultured with iPSC-MSCs-EVs.
As shown in fig. 6A, in the control group, a large number of normal follicles were observed in the ovaries at different periods. The number of primordial and primary follicles was significantly reduced in the mice of the CTX group, but in the CTX-EV group, the number of primordial follicles was significantly increased compared to the CTX group, and the number of primary follicles was also significantly increased. The follicle count results (fig. 6B) further demonstrate a statistically significant reduction in primordial and primary follicle numbers in the CTX group of mice, in contrast. However, in the CTX-EV group, the number of primordial follicles was significantly increased compared to the CTX group, and the number of primary follicles was also significantly increased. There were no significant differences between the three groups for secondary versus mature follicles.
The immunohistochemical experiment result (figure 6C) also proves that the iPSC-MSC-EVs can reduce the expression of an apoptosis marker, namely cleaned Caspase 3, and increase the expression of a cell proliferation marker, namely Ki 67.
The result shows that iPSC-MSC-EVs can inhibit apoptosis caused by chemotherapy by adjusting ILK signal channel and promote the recovery of ovaries.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of organic chemistry, polymer chemistry, biotechnology and the like, and it is understood that the present invention may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Many modifications and variations are possible in light of the above teaching and are therefore within the scope of the invention.
In the present invention, "about" means ± 10%, preferably ± 5%, more preferably ± 2%, for example, ± 1%, ± 0.5% or ± 0.1%.
Claims (12)
1. A method of modulating an ILK signaling pathway in a cell comprising administering to the cell an exosome inducing pluripotent stem cell-derived mesenchymal stem cells,
for example, it is a method for modulating the ILK signaling pathway in vitro cultured cells, which comprises adding to the cell culture broth an exosome derived from mesenchymal stem cells inducing omnipotent stem cell origin.
2. The method of claim 1, wherein the mesenchymal stem cells are cells that have been passaged 1-15 times, preferably 1-10 times, most preferably 3-7 times.
3. The method according to claim 2, wherein the amount of exosomes added is about 1-500ug/ml, preferably about 5-250ug/ml, more preferably about 10-200ug/ml.
4. The method of any one of the preceding claims, wherein the cell is a cell with an aberrant ILK pathway (e.g. a down-regulated ILK activity).
5. A method according to any preceding claim, wherein ILK pathway activity of the cell is restored, for example such that the cell ILK activity is upregulated.
6. Use of inducing a universal stem cell-derived mesenchymal stem cell-derived exosome for the preparation of a medicament for the treatment of an ILK signalling pathway related disease, preferably wherein the mesenchymal stem cell is a cell passaged 1-15 times, preferably 1-10 times, most preferably 3-7 times.
7. The use according to claim 6, wherein about 1-500ug, preferably about 5-250ug, more preferably about 10-200ug of exosomes are administered to the patient.
8. The method according to claim 6 or 7, wherein the ILK signaling pathway-related disease is an abnormal or pathological angiogenesis-related disease (e.g., cancer or heart disease), a metabolic disorder, an inflammatory disease, or an ovarian-related reproductive disorder.
9. A pharmaceutical composition for treating an ILK signaling pathway-related disease such as an abnormal or pathological angiogenesis-related disease (e.g., cancer or heart disease), a metabolic disorder, an inflammatory disease, or an ovary-related reproductive disorder, comprising inducing multipotent stem cell-derived mesenchymal stem cell-derived exosomes.
10. The pharmaceutical composition according to claim 14, wherein the exosome is dosed at about 1-500ug, preferably about 5-250ug, more preferably about 10-200ug.
11. The pharmaceutical composition according to claim 9, which is in the form of a single or multiple administration, preferably in the form of multiple administrations, such as in the form of administrations over about 1-7 days.
12. The pharmaceutical composition according to claim 11, wherein each administration dose is about 1-500ug, preferably about 5-250ug, more preferably about 10-200ug,
for example, from about 40 to about 5000ug/kg body weight, preferably from about 400 to about 4000ug/kg body weight.
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